This project addresses a major barrier in designing HIV-1 entry inhibitors that stems from the high conformational variability of the viral envelope glycoprotein gp120. Diverse experimental observations have shown that the HIV-1 envelope protein undergoes large conformational structuring upon binding to its host cell receptors. These conformational changes lead to progressive maturation of Env binding sites for the cell receptors and are required for virus-cell fusion, cell entry and consequent infection. In addition, several types of Env inhibitor leads have been identified that appear to function by binding the unliganded state of HIV-1 gp120 and conformationally entrapping Env protein into states that have suppressed receptor binding site activity for cell entry. This body of observations argues that knowledge of differences in the locations and dynamics of conformational transitions of unliganded gp120 induced by receptors vs inhibitors could help to identify inhibitors that most effectively frustrate the former while encouraging the latter. Yet, the unliganded state itself, which is in essence the primary binding target for antagonists of viral envelope, is poorly understood, as are the dynamics of conformational changes that occur starting from this structure. These shortcomings will be addressed in the current R21 project by using innovative strategies to track site-specific conformational changes leading from unliganded to liganded forms of Env gp120. The R21 project will have two specific aims. In Aim 1, we will produce fluorophore-tagged gp120 variants in which the Env protein is labeled in different structural locations predicted to change conformations upon binding to known envelope ligands, including CD4 and both CD4-mimicking and allosteric inhibitors. In Aim 2, we will measure location- specific changes in both time-resolved and steady state fluorescence anisotropy upon ligand binding to single- site labeled gp120 variants. Experimental anisotropy curves derived from distinctly labeled sites will be used to define conformational signatures of gp120 in unliganded, activated and inhibited states. The most immediate outcomes of the R21 project will be (1) derivation of functionally intact gp120 variants with location-specific fluorophore reporter tags and (2) demonstration that ligand-induced conformational changes can be detected at spatially defined sites in gp120 through fluorescence tracking. Longer term research following up from the R21 will map specific sites of conformational changes in gp120 that occur upon receptor activation vs antagonist (competitive as well as allosteric) inhibition and derive fluorescence-based screens for inhibitors that induce specifically localized conformational changes leading to improved inhibition of HIV-1 Env protein. PUBLIC HEALTH RELEVANCE: This project will establish an innovative multidisciplinary approach, combining protein engineering and fluorescence spectroscopy, to expand understanding of the structural transitions of HIV-1 virus coat protein and long term guide identification of entry inhibitors as antiviral agents.